Cardiovascular disease is among the leading causes of morbidity and mortality. Plasma estrogen concentration is closely related to cardiovascular health in a woman's life, and menopause is associated with a substantial increase in the risk of cardiovascular disease. However, the mechanisms underlying estrogen's effects are still far from being completely understood. The novel G protein- coupled estrogen receptor 1 (GPER) has been demonstrated to participate in many cardiovascular functions. Calmodulin (CaM) is involved in virtually all aspects of cell functions but is not expressed sufficiently for all its targets. Factors that alter the expression of total and free CaM potentially have profound implications for cardiovascular functions. In this application we propose to test the overall hypothesis that GPER activation triggers in the vasculature feed-forward mechanisms that involve CaM both at the receptor level and downstream effectors. Three specific aims are proposed. Aim 1 will identify GPER as a novel CaM-binding protein. The CaM-binding domain in GPER will be identified, GPER CaM interactions will be characterized, and a biosensor to mirror GPER's CaM-dependent activities will be generated. Aim 2 will test the hypothesis that in the vasculature, GPER activation triggers feed-forward mechanisms in which total and free CaM expression is upregulated, which facilitates the activities of GPER and the classical estrogen receptor ER1 as CaM-binding proteins and links signaling pathways mediated by these two receptors. Aim 3 will examine the effects of GPER activation on endothelial Ca2+ signaling and cell-cell interaction via modulation of CaM binding and activity of the plasma membrane Ca2+-ATPase (PMCA), the platelet-endothelial cell adhesion molecule (PECAM-1), and the potential interaction between PECAM-1 and sphingosine 1 phosphate receptor 1 (S1P1). Our long-term goal is to understand the mechanisms of action and roles of GPER in CaM- dependent activities in the cardiovascular system. Data from these studies will enhance our understanding of how estrogen works and provide timely information on the regulatory mechanisms and effects of activation of GPER in the vasculature, which will give further basis for options to target estrogen receptor subtypes for preventive and therapeutic purposes. PUBLIC HEALTH RELEVANCE: Despite the clear linkage between post menopause and cardiovascular disease, hormone replacement therapy has not proven to be cardio protective. This application will identify the novel G protein-coupled estrogen receptor 1 (GPER) as a novel calmodulin-binding protein and test the overall hypothesis that GPER activation triggers in vascular endothelial cells and smooth muscle cells feed-forward mechanisms that involve CaM both at the receptor level and downstream effectors, linking signaling pathways mediated by different estrogen receptors. Effects of GPER activation will also be tested on endothelial Ca2+ signaling and cell-cell interaction via modulation of calmodulin-dependent activities. It is expected that the proposed studies will provide timely information that help form the basis for targeting estrogen receptor subtypes for preventive and therapeutic purposes.